The present invention relates to a reagent kit for detecting luminescence (light emission) from luciferin/luciferase reactions which is characterized by the use of an organic sulfur regent, and to a method for detecting a Coleoptera luciferase using the kit.
The detection kit provided according to the present invention is applicable to any assay system in which a Coleoptera luciferase enzyme is used as a reporter or signal.
In in vitro luciferin/luciferase luminescent reactions, a flash of light is observed as an emission pattern. Therefore, it has been impossible to determine such luminescent reactions precisely unless a specialized device that allows the injection of a measured amount of a luminescent reagent. To overcome this problem, a method for luciferase detection has been invented which uses a thiol reagent so that the half-life of the luminescence can be extended to about five minutes with increased quantity of light emission(Japanese Patent No. 3,171,595). According to this method, the quantity of light emitted from a luciferin/luciferase reaction can be determined precisely with a luminometer or liquid cintillation counter even if they do not have an automated luminescent reagent injection device. Since this method was invented, it has become possible to achieve sensitive determination of a luciferase expressed in cultured cells or the like by using a luciferase gene as a reporter gene. Thus, luciferin/luciferase luminescent reactions have been widely utilized.
However, the thiol reagents have unpleasant odor originated from a mercaptan residue, and the efficiency of measurement operation is poor. Dithiothreitol (DTT), which is described as a thiol regent in the patent publication above, is a very expensive reagent and, moreover, should be used in a high concentration of 5 mM or higher for achieving sufficient luciferin/luciferase reactions that measurement can be performed without any trouble. It is also known that DTT tends to be degraded readily in such a high concentration solution as described in the above patent publication and the DTT oxidized or degraded by dissolved oxygen or the like significantly reduces the efficiency of luciferin/luciferase reactions.